Enzymatic resolution of racemic (cis-1,2-epoxypropyl) phosphonic acid compounds

ABSTRACT

Enantiomeric mixtures of esters and amides of (cis-1,2epoxypropyl)phosphonic acid (-) stereoselectively hydrolyzed by enzymes of micro-organisms to produce (-) (cis-1,2epoxypropyl)phosphonic acid. This product and its salts have antibacterial activity.

United States Patent Demain et a].

[ 1 Jan. 18, 1972 Raymond F. White, Englishtown, all of NJ.

A Assignee: Merck 8: Co., lnci, Rahway, NJ.

Filed: Dec. 27, 1968 Appl. No.: 787,580

US. Cl ..195/2, 195/29, 195/30 Int. Cl. ..Cl2d 9/00 Field of Search..195/2, 29, 30, 80, 114

References Cited FOREIGN PATENTS OR APPLICATIONS 1,046,047 12/ 1958Germany ..424/203 1,087,066 10/ 1967 Great Britain ..424/203 OTHERPUBLICATIONS Churi et aL, J. ofAm. Chem. Soc., 83,1966, pp. 1824-1825Chemical Abstracts, 62 1'965), l0457(f) Derwent Farmdoc No. 35,893,Abstracting BE718,507, Publ. l-2469 Primary Examiner-Joseph GolianAttomey-John Frederick Gerkens, J. Jerome Behan and I. Louis Wolk [57]ABSTRACT Enantiomeric mixtures of esters and amides of(cis-1,2-epoxypropyl)phosphonic acid stereoselectively hydrolyzed byenzymes of micro-organisms to produce (cis-l,2-epoxypropyl)phosphonicacid. This product and its salts have antibacterial activity.

l2 Clairns, No Drawings 1: ENZYMATIC RESOLUTION OF RACEMIC (ClS-l,2-EPOXYPROPYL) PHOSP'IIONIC ACID COMPOUNDS This invention is concernedwith a method of separating (cis-l,2-epoxypropyl)phosphonic acid fromenantiomeric mixtures of derivatives of (cis-l,2-epoxypropyl)phosphonicacid. More particularly, it relates to a method of stereoselectivelyhydrolyzing esters and amides of (cis-l,2-epoxypropyl )phosphonic acid.

BACKGROUND OF THE INVENTION (cis-l,2-epoxypropyl)phosphonic acid and itssalts are valuable antibiotics which are effective against variousgrampositive' and gram-negative pathogens. Although these antibioticscan be produced by fermentation of suitable strains of Streptomyces, forexample by growing Streptomyces fradiae NRRL B-335l aerobically insuitable fermentation mediums, other methods have been found whereby theantibiotic, its salts, esters, and amides, can-be prepared by synthesis,for example, from cis-propenylphosphonic acid and derivatives thereof.One disadvantage of such synthetic methods is that the desired productis obtained mixed with.its enantiomer, usually in the form of aracemate, and it must be separated in order to obtain the product inpure form.

SUMMARY OF THE INVENTION It is an object of the present invention toprovide a method whereby the isomer of (cis-l,Z-epoxypropyl)phosphonicacid can be obtained from mixtures of esters and amides of and(cis-l,2-epoxypropyl)phosphonic acid. Another object is to provide aprocess for stereoselectively hydrolyzing esters and amides of(cis-1,2-epoxypropyl)phosphonic acid stereoisomers to obtain(cis-l,2-epoxypropyl)phosphonic acid and salts thereof. Other objectswill be apparent from the detailed description of this inventionhereinafter provided.

In accordance with the present invention, it is found that ester andamide antipodes of (cis-l,2-epoxypropyl)phosphonic acid can beselectively hydrolyzed by subjecting such mixtures to the action ofenzymes of selective hydrolyzing strains of micro-organisms to producemixtures containing (cis-l,2-epoxypropyl)phosphonic acid or saltsthereof. In carrying out the process of this invention, the esters oramides can be added to a nutrient medium in which the micro-organism isgrown, or intimately contacted with cells of the organism in suitablebuffered mediums.

Thus, in accordance with preferred embodiments of this invention, whenantipode mixtures of esters and amides of the formula wherein Xrepresents OR or NR,R and Y represents NR,R or H where R is a straightorbranched-chain lower alkyl, lower alkenyl, or lower alkynyl group and R,and R represent the same or different hydrocarbyl groups, substitutedhydrocarbyl groups or hydrogen, and are subjected to the action ofenzymes of certain micro-organisms the esters and amides of(cis-l,2-epoxypropyl)phosphonic acid are selectively hydrolyzed toproduce the phosphonic acid without concomitantly hydrolyzing the(cis-l,2-epoxypropyl)phosphonic acid derivatives. The(cis-l,2-epoxypropyl)phosphonic acid or a salt thereof can then beseparated from the esters or amides of the enantiomer.

Pursuant to one preferred embodiment of thisinvention, monoesterenantiomeric mixtures of compounds of the formula and salts thereofwherein R is a straightor branched-chain alkyl groupofone to sevencarbon atoms, analkenyl group of two to seven carbon'atoms, or an alkylgroup of two or seven carbon atomsare especially useful startingmaterials since the enantiomer can be cleaved substantiallyquantitatively without concomitant hydrolysis of the enantiomer, therebyresulting in a mixture of (cis-l,2-epoxypropyl)phosphonic acid'and themonoester which can be readily separated to obtain the desired acid or asalt thereof.

Similarly, enantiomeric mixtures of amides of the general formulawherein R, and R are the same as defined above and 2 represents NR,R orOH, or salts thereof can be selectively hydrolyzed to obtain(cis-l,2-epoxypropyl)phosphonic acid while the amides remainsubstantially unchanged. Thus, the (cis-l,2-epoxypropyl)phosphonic acidin the resulting reaction mixture can be separated from the amide of(cis-l ,2-epoxypropyl)phosphonic aicd.

'In the foregoing structural formula of the amide, the substituents R,and R, can be aliphatic, cycloaliphatic araliphatic,

.aromatic or heterocyclic radicalswhich can, if desired, be

further substituted. Thus, for example, it can be aliphatic such assubstituted or unsubstituted alkyl, alkenyl or alkynyl, representativeexamples of which are alkyl such as methyl, propyl, isopropyl, t-butyl,hexyl, octyl, decyl, dodecyl, haloalkyl such as chloroethyl,fluoropropyL'bromomethyl and dichloroethyl, acylarnidoalkyl such asacetylaminomethyl and benzoylaminoethyl, acyloxyalkyl such asacetoxymethyl, propionoxyethyland benzoyloxyethyl, other substitutedalkyl groups such 7 as hydroxypropyl, piperidinomethyl, aminomethyl andaminoethyl, alkylaminoalkyl such as dimethylaminomethyl,diethylaminopropyl, carboalkoxymethyl, cyanoethyl, sulfonamidoethyl,phthalimidomethyl and methoxymethyl; alkenyl such as allyl, methallyl,vinylpropenyl, hexenyl, octadienyl, alkynyl such as propargyl, ethynylon chlorethynyl; cycloalkyl such as cyclohexyl, cyclohexenyl orcyclopropyl. When R, and R are aliphatic, they preferably have from oneto sixcarbon atoms.

Examples of R, and R representing an araliphatic radical are those caseswhere it is aralkyl such as benzyl, phenethyl, phenylpropyl,p-halobenzyl and o-, mor p-alkoxybenzyl, nitrobenzyl, aminophenethyl,pyridylethyl, furylmethyl, thienylpropyl and the like.

R, and R may also represent an aryl radical such as phenyl, naphthyl orsubstituted phenyl, e.g., p-chlorophenyl, onitrophenyl,o,p-dihalophenyl, cyanophenyl, methoxyphenyl, aminophenyl andtolyl, andpreferably a mononuclear aromatic residue. vWhen R, or R isheterocyclic, it can be heteroaromatic such as' pyridyl, furyl, thienyl,thiazolyl or pyrazinyl, or altematively it can represent a hydrogenatedhetero ring, examples of which are tetrahydrofuryl and piperazinyl.

When- R, and/or R are acyl, they are preferably lower alkanoyl or aroylsuch as acetyl, propionyl, butyryl, hexanoyl,

'benzoyLthalobenzoyl, nitrobenz'oyl and the like.

In accordance with the present invention, it is found that variousmicroorganisms producing enzymes capable of selectively hydrolyzingesters and amides of (cis-l ,Z-epoxypropyl)phosphonic acid areavailable. Thus, bacteria such as those of order Actinomycetes, forexample Nocardia corallina;

fungi such as suitable Penicillium strains, for example Penicilliumfrequentans and'Penicillium vermiculatum; Aspergillus strains, forexample Aspergillus niger Fungi imperfecti, for example Ashbya'gossypiiand Helicostylum pinfarme have all been found to produce enzymes capableof selectively hydroylzing' esters and amides of(cis-l,2-epoxypropyl)phosphonic acid.

The micro-organism producing useful enzymes for carrying out theprocesses of this invention can readily be found either by cultivatingknown micro-organisms or unknown micro-organisms obtained from sourcessuch as soils and the like in the presence of a monoester or amide of(cis-l,2-epoxypropyl)phosphonic acid and determining after good growthof the organism is achieved (2-5 days usually being sufficient for thispurpose) whether or not the supernatant liquor has activity asdetermined by the Proteus vulgaris assay. Alternatively, this samescreening can be effected using resting cells containing the enzyme andincubating such cells in suitable media such as, for example, Tris 0.05Mbuffer at pH 8.0 for 24 days and then assaying the liquid portion withProteus vulgaris. In carrying out such tests it is desirable tosimultaneously inoculate the test micro-organism in a suitable nutrientmedium without the monoester or amide in order to distinguish thoseorganisms which produce the antibiotic directly from those capable ofselectively hydrolyzing the esters or amides.

The following examples illustrate methods of carrying out thisinvention.

EXAMPLE 1 A medium containing 0.8 percent nutrient broth (Difco), 0.2percent yeast extract, 3 percent cerelose and 0.3 percent malt extractin distilled water is adjusted to pH 7.0. 40 ml. of this medium isplaced in a 250 ml. Erlenmeyer flask which is then autoclaved at psi.for 15 minutes. The medium is then inoculated with a loopful of inoculumfrom an agar slant of Aspergillus niger NRRL-67 and incubated on amechanical shaker operating at 220 r.p.m. with a 2-inch throw at 28 C.for 4 days until good growth of the organism is obtained. Ten ml. of theresulting fermentation broth is aseptically transferred to a centrifugetube and the cells are pelleted in a centrifuge at 25,000XG. Thesupernatant fluids are discarded and the pelleted cells are resuspendedin 4 ml. of 0.05 M (hydroxymethyl)-aminomethone buffer (Tris buffer)adjusted to pH 8.0. Two ml. of the resulting cell suspension is thenaseptically transferred to a sterile X200 mm. test tube containing 200of sodium monomethyl racemic (cis-l,2-epoxypropyl)phosphonate in 2 ml.of 0.005 (hydroxymethyl)- aminomethane pH 8.0 buffer. The tube isincubated for 48 hours at 28 C., on a mechanical shaker. The resultingincubated broth is centrifuged at 25,000 G and the supernatant fluid isexamined for biological activity by disc assay with Proteus vulgarisMR-838 (ATCC 21100 and NRRL B4361) and found to have an inhibition zoneof 26 mm. indicating the presence of a salt of(cis-l,2-epoxypropyl)phosphonic acid.

Control tubes containing (1) 2 ml. of cell suspension plus 2 ml. of 0.05M (hydroxymethyl)-aminomethane pH 8.0 buffer and (2) 4 ml. of the samebuffer containing 2007 of sodium monomethyl racemic(cis-l,2-epoxypropyl)phosphonate are similarly incubated for 48 hr. at28 C. The supernatant liquid from each of these control tubes is foundto give no inhibition in the Proteus vulgaris assay.

The assay using Proteus vulgaris MB-838 is carried out as follows.

The test culture is maintained as a slant culture on nutrient agar(Difco) plus 0.2 percent yeast extract (Difco). The inoculated slantsare incubated at 37 C. for 18-24 hours and stored at refrigeratortemperatures for 1 week, fresh slants being prepared each week.

The inoculum for the assay plates is prepared each day by inoculating a250 ml. Erlenmeyer flask containing 50 ml. of nutrient broth (Difco)plus 0.2 percent yeast extract (Difco) with a scraping from the slant.The flask is incubated at 37 C. on a shaking machine for 18-24 hours.The broth culture is then adjusted to 40 percent transmittance at awavelength of 660 mu using a Bausch & Lomb Spectronic 20 by the additionof 0.2 percent yeast extract solution to the growth. Uninoculated brothis used as a blank for this determination. 30 ml. of the adjusted brothis used to inoculate 1 liter of medium.

Nutrient agar (Difco) plus 0.2 percent yeast extract (Difco) is used asthe assay medium. This medium is prepared, sterilized, by autoclaving,and allowed to cool to 50 C. After the medium is inoculated, [0 ml. isadded to sterile petri dishes and the medium is allowed to solidify.

Samples of the supernatant liquid to be assayed are diluted in 0.05 MTris buffer at pH 8.0 to an appropriate concentration. Discs are dippedinto the test solution and placed on the surface of the assay plate; twodiscs for each sample are normally placed on one plate opposite to oneanother. Two discs dipped into 0.4 units per ml.(cis-l,2-epoxypropyl)phosphonic acid solution are placed on the plate inan alternate position to the sample. The plates are incubated at 37 C.for 18 hours and the zone diameters in millimeters are determined. Thepotency of the sample is determined by means of a nomograph or from thestandard curve. One mg. of pure (cis-l,2-epoxypropyl)phosphonic acidcontains 357 units, a unit being as the concentration of the productwhich will produce a zone diameter of 28 mm.

EXAMPLE 2 When the procedure of example I is repeated using Ashbyagossypii NRRL Y 1056 in place of Aspergillus niger the supernatantliquid is found to have an inhibition zone of 18 mm. in the Proteusvulgaris assay.

EXAMPLE 3 When the procedure of example 1 is repeated using 2007 ofsodium monoethyl racemic (cis l ,2-epoxypropyl)phosphonate, theresulting supernatant liquid is found to give a 30 mm. zone ofinhibition upon assay with Proteus vulgaris.

EXAMPLE 4 Following the procedure of example I using the same quantityof the sodium salt of the monopropyl racemic ester in place of themonomethyl ester, the resulting supernatant liquid is found to give aninhibition zone of 20 mm. in the Proteus vulgaris assay. EXAMPLE 5Following the procedure of example 1 but substituting the same quantityof the sodium salt of the monoallyl racemic ester in place of themonomethyl ester, the resulting supernatant liquid is found to give a 30mm. zone of inhibition upon assay with Proteus vulgaris.

EXAMPLE 6 Following the procedure described in example 1, resting cellsof Ashbya gossypii NRRL Y 1056 are obtained in pellet form. When thesecells are incubated in Tris buffer at pH 8 with 2 ml. of a 200y per ml.solution of sodium monoethyl racemic (cis-l,2-epoxypropyl)phosphonateusing the procedures described in example 1, the supernatant liquorobtained after incubation for 48 hours is found to give an inhibitionzone of 1 mm. by the Proteus vulgaris assay.

EXAMPLE 7 Following the procedures described in example 6 butsubstituting the sodium salt of the monoallyl racemic ester in place ofthe monoethyl ester, it is found that the resulting incubated broth hasan inhibition zone of 24 mm. as determined by the Proteus vulgan'sassay.

EXAMPLE 8 Following the procedures described in example I, resting cellsof Nocardia corallina ATCC 4273 are obtained in pellet form. When theseresting cells are incubated with the sodium salt of the monomethyl esterof racemic (cis-l ,2-epoxypropyl)phosphonic acid, following theprocedures described in example 1, the resulting incubated broth isfound to have an inhibition zone of 12 mm. as determined by the Proteusvulgaris assay.

EXAMPLE 9 When the procedure of example 8 is repeated using the samequantity of the sodium monoethyl ester salt in place of the monomethylester salt, the resulting incubated broth is found to have an inhibitionzone of 13 mm. as determined by the Proteus vulgaris.

EXAMPLE When the procedures of example 8 are repeated using the samequantity of the sodium monopropylracemic estersalt in place of themonomethyl racemic estersalt, the resulting incubated broth is found tohave an inhibition zone of 11 mm. as determined by the Proteus vulgarisassay.

EXAMPLE 11 When the procedures of example 8 are repeated using the samequantity of the sodium monoallyl racemic estersalt in place of themonomethyl racemic ester salt, the resulting incubated broth is found tohave inhibition zone of 19 mm. as determined by the Proteus vulgarisassay.

EXAMPLE 12 Following the procedures described in example 1, Pencilliumfrequentans NRRL 1915 is cultured and the resulting cells isolated as apellet. The resting cells are then incubated with the sodium salt ofmonomethyl racemic (cis-1,2-epoxypropyl)phosphonic acid and theresulting liquid solution assayed by means of the Proteusvulgarisprocedures. The assay shows that the product has an inhibitionzone of 21 mm.

EXAMPLE 13 To 40 ml. of a sterile medium consisting of 0. 8 percentnutrient broth, 0.2 percent yeast extract, 3 percent cerelose and 0.3percent malt extract in distilled water adjusted to pH 7.0 in a 250 ml.Erlenmeyer flask is added-inoculum of Aspergillis niger NRRL 67 from anagar slant. The inoculated flask 50incubated C., mechanical shakerrunning at 220 r.p.m. with a 2-inch throw at 28 C. for 4 days. Ten'ml.of the resulting fermentation broth is then aseptically transferred to acentrifuge, and the cells are pelleted at 25,000XG. The supernatantliquid is discarded, and the pelleted cells are resuspended in 4 ml. of0.05 Tris buffer at pH 8.0. Two ml. of the resulting cell suspension isthen aseptically transferred to a sterile X200 mm. test tube containing200 of N,N'-tetraethyl racemic (cis-l,2-expoxypropyl)phosphonamide in 2m1. of 0.05 M Tris buffer at pH 8.0. The tube is then incubated for 48hours at 28 C. in a mechanical shaker running at220 r.p.m. with a 2-inchthrow. After incubation the cells are removed by centrifugation and thesupernatant liquid assayed for biological activity with Proteusvulgart's MB-838. The assay showed the solution to have aninhibitionzone of 38 mm.

EXAMPLE 14 Following the procedures described in example 13,Helicastylum pinforme ATCC 8992 is grown and the resulting cells areincubated with N,N'-tetramethyl racemic (cis-l,2-epoxypropyl)phosphonamide for 2 days at 28 The-supernatant liquid oftheresulting incubated broth is found to give an inhibition zone of mm.when assayed with Proteus vulgaris MB-838.

EXAMPLE 15 Following the procedures described in example 13, cells ofPencillium frequentans NRRL 1915 are incubated with N,N- tetramethylracemic (cis-1,2-epoxypropyl)phosphonamide and the resulting incubatedfluid portion is found tohave an inhibition zone of 39 mm. when assayedwith Proteus vulgaris.

EXAMPLE 16 When resting cells of Aspergillusfumigatus NRRL 165 are usedin place of Aspergillus niger in example 13, the supematant fluidis'found to give a 44 mm. zone of inhibition when assayed with Proteusvulgaris MB 838.

EXAMPLE l7 Using'the procedures described in example 13, resting cellsof Pencillium vermiculatum, culture 54 B in Quatermaster collection atNatick, Conn., are incubated with N,N' tetramethyl racemic(cis-l,2-epoxypropyl)phosphonamide for 48 hours and'the resultingsupernatant liquid is found to have an inhibition zone of 32mm. whenassayed with Proteus vulgar-is MB 838.

EXAMPLE .18

.A solution of 1.2 g. KH PO 2.4 g. Na i-l P0 0.4 g. Nl-LCl, 0.002 g.2nSO -5H O, 0.004 g. FeSO 2 g. CaCO; and 2 g. of ethyl lactate in 400ml.of tap water in a 4-liter Erlenmeyer flask-is adjusted to pH 7.0 and theflask sterilized by autoclaving at 121C. and I5 p.s.i. for 15 minutes.To the resulting sterile medium is added a vegetative growth ofAspergillus niger NRRL 67 and the resulting inoculatedflask is incubatedfor 4 days on a rotary shaking machine operating at 220 r.p.m. with a2-inch throw at 28 C. To this fermenting medium is then added 40 ml.ofan aqueous solution containing 20 g. of

sodium monoethyl racemic (cis-l ,2-epoxypropyl)phosphonate, previouslyneutralized to 'pH 7 and sterilized by filtration. The incubation isthen continued for another 24 hours.

The resulting fermentation medium -is then filteredand the filtrate isrepeatedly evaporated with methanol in vacuum until a solutionconsisting of *percent methanol is obtained. This methanol solutionisthen filtered to remove precipitated inorganic .salts and the residuewashed withmethanol which is added tothe filtrate. The resultingmethanol solution is chromatographed ona column containing 2,000 g. ofalumina; the concentrated solution being added to :thealumina'previously washed with methanol. The .column is then developedwith methanoland 200ml. fractionsof eluate are taken. The fractions areassayed by means of the Proteus vulgaris assay procedure,'and thefractions containing antibiotic activity are combined and the methanolremoved by evaporation under diminishedpressureThe residue containingthe sesqui sodium salt of (cis-1.;2-epoxypropyl)phosphonic from aqueousmethanol to obtain :the sesqui sodium'salt in pure crystalline form.- A2 .percent solution of this product in water has a pH of 6.8 and an The.enantiotneric esters and amides of (cis-l,2epoxypropyl)phosphonic acidused in the "process of this invention are obtained by epoxidizing thecorresponding derivatives of cis-propenylphosphonic acid.

Thus,the monoesters-are prepared by .epoxidizing the correspondingmonoesters of cis=monopropenylphosphonic acid or, alternatively, by.acid hydrolysis .of the diester of (cis-l,2- epoxypropyl)phosphonicacid. For example, the monoesters can be prepared starting with dit=butyl-phosphorochloridite as follows:

' Phosphorus trichloride (68.7a-grams, 0.5 mole) and 750 ml. ofanhydrous benzeneare placed in a 2-liter, 3-necked flask equipped withmechanical stirrer,-thermometer, dropping funnel and a drying tube. Thesolution is cooled to 5 C. and triethylamine (50.6 grams,'0.5 mole) isadded at 5-l0 C. during 20 minutes. The benzene solution is then stirredfor 20 .minutes. A solution of triethylamine (50.6 grams, 0.5 mole) and.t-butanol (37.06 grams, 0.5 mole) is added with stirring during 20minutes at "5-10 C., and the mixtureis stirred for 20 minutes. A secondportion of t-butanol (37.06 grams, 0.5 mole) is then added at .5-' 1 0C. overa 20 minute period and the reaction mixture containing di-t-butylphosphorochloridite is stirred'for 90'minutesat 5-l0 C.

between and C. by external cooling. The resulting mix-.

ture containing di-t-tubyl-2-propynylphosphite is stirred at 5- 10C. for1 hour.

The reaction mixture containing di-t-butyl-2-propynylphosphite is thenheated to reflux, and the refluxing is continued for 1 hour. Thesolution is then cooled to room temperature with a water bath, and 185ml. of water is added in portions. The triethylamine hydrochloridedissolved in the aqueous layer, and the organic layer is separated fromthe aqueous layer. The benzene solution is heated at atmosphericpressure, and the water is removed by azeotropic distillation. Theresidual benzene solution contains di-t-butylpropadienylphosphonate.

Where desired, the pure ester can be obtained by removing the solventand distilling the ester under high vacuum; the pure ester ischaracterized by IR and NMR spectra.

The dried benzene solution of di-t-butyl-propadienylphosphonate ishydrogenated at 25 C. with 5 percent palladium-on-carbon catalyst (5.0grams) until hydrogen absorption ceases. The catalyst is removed byfiltration and is washed with 2X50 ml. of benzene. Upon removal of thesolvent in vacuo, di-t-butyl-cis-propenylphosphonate is obtained and ischaracterized by NMR and IR spectra. The crude ester can be purified byhigh-vacuum distillation.

Di-t-butyl-cis-propyenylphosphante (1.0 mole) and paratoluene sulfonicacid (0.005 mole) are dissolved in 235 ml. of benzene, and the solutionis heated at reflux until the calculated amount of isobutene is formed.A gas metering device is employed to detect the isobutene. The reactionmixture is then cooled to room temperature, the solvent is removed invacuao and cis-propenylphophonic acid is obtained as the residue.

The diesters of cis-propenylphosphonic acid are obtained by firstconverting the free acid to cis-propenylphosphonic dichloride, and thenreacting this dichloride with 2 molar equivalents of an alcoholrepresented by the formula R-OH, where R represents the alcohol residueof the resulting ester. The monoesters are prepared from the diesters byremoval of one of the ester radicals with base. A monosalt monoester maythen be prepared by reacting the monoester with one equivalent of base.Representative examples for the foregoing reactions are given herein,and it is to be understood that other esters and salts are obtained inthe same manner from the appropriate starting materials.

a. Into a 250 ml. three-necked round-bottomed flask there is added 6.1g. of cis-propenylphosphonic acid, 60 ml. of dry benzene and 9.0 ml. ofpyridine. The mixture is heated to 50 C., the heat removed and 13.2 g.of thionyl chloride added dropwise at such a rate as to keep thereaction mixture at 50 C. The mixture is then cooled to room temperatureand stirred for 2 hours at room temperature. The mixture is filtered andthe filtrate concentrated in vacuum at 35 C. to yield 4.5 g. of a turbidoil. It is distilled to afford cis-propenylphosphonic dichloride, b.p.67-69C./910 mm.; n 1.4885.

b. A stirred mixture of 0.1 mole of cis-propenylphosphonic dichlorideand 0.2 mole of triethylamine in 100 ml. of benzene is cooled to 5 C. Tothe mixture there is added 0.2 mole of methyl alcohol at such a rate asto maintain the temperature at 510 C. When the addition is complete, themixture is stirred at room temperature for 1 hour. The precipitatedtriethylamine hydrochloride salt is then filtered ofi and the solvent isremoved at reduced pressure to leave dimethyl cispropenylphosphonate.Other diesters of cis-propenylphosphonic acid such as the alkyl,alkenyl, alkynyl, substituted alkyl, alkenyl or alkynyl esters areprepared in the same manner using 0.2 mole of the appropriate alcohol.

The dimethyl cis-propenylphosphonate is then converted to the sodiumsalt of the monomethyl cis-p-propenylphosphonate heating the diester inaqueous sodium hydroxide for sufficient time to convert the diester tothe monoester. Similarly, other dialkyl, dialkenyl, or dialkynyl estersare' converted to the salt of the monoester in the manner.

c. To a 50 ml. round-bottom 3-necked flask fitted with a stirrer,thermometer and addition funnel is added 1.5 g. (0.01 mole) of sodiummonomethyl cis-propenylphosphonate in 30 ml. of methanol. The pH isadjusted to 4.5 with 2.5 N sodium hydroxide. Sodium tungstate dihydrate(0.06 g.) is added and the mixture heated to 55 C. Hydrogen peroxide 30percent (3 ml.) is added dropwise over 15 minutes at 55-60 C. Hydrogenperoxide is added as needed to maintain a positive starch-iodide test.The mixture is quenched by adding saturated sodium sulfite solution to anegative starch-iodide test. The inorganic solids are removed byfiltration, the solution evaporated to one-half volume and chilled. Thesolid sodium methyl (i) (cis-1,2-epoxypr0pyl)phosphonate thus obtainedis separated by filtration, washed with ethanol and dried. The sameresult is obtained with zinc vandate or sodium selenomolybdotungstate isused as cataylst in place of sodium tungstate.

When an equimolar amount of sodium n-butyl cis-propenylphosphonate,sodium ethyl cis-propenylphosphonate, sodium monovinylcis-propenylophosphonate, potassium monoallyl cis-propenylphosphonate orpotassium propargyl cispropenylphosphonate is expoxidized as describedabove, the sodium salt of the n-butyl, ethyl, vinyl, ally], andpropargyl ester of racemic (cis-l,Z-epoxypropyl)phosphonate is obtained.

The phosphono amides and diamides employed as starting materials areobtained by reacting the cis-propenylphosphonic dichloride with aprimary or secondary amine. When one mole of amine is used, one of thechlorides is displaced to afford a cis-propenylphosphonochloridic amide.

The diamides are obtained from the cis-propenylphosphonic dichlorideaccording to the above procedures by utilizing 2 moles of primary orsecondary amine. The monoamides or diamedes of cis-propenylphosphonicacid so obtained can then be reacted with hydrogen peroxide to producethe corresponding amides of (cis-1,2-epoxypropyl)phosphonic acid. Forexample, racemic N,Ndibenzoyl (cis-l,2-epoxypropyl)phosphondiamide isprepared as follows.

To a mixture of 3.25 g. of N,N-dibenzoyl-cis-propenylphosphonodiamide in15 ml. of methanol there is added 0.06 g. of sodium tungstate dihydrateand the mixture heated to 55 C. Three ml. of 30 percent hydrogenperoxide is then added slowly over 15 minutes, and the resulting mixturestirred for 1 hour at 55 C. The hydrogen peroxide is added during thistime as necessary to maintain a positive starchiodide test. At the endof the 1 hour, saturated aqueous sodium sulfite is added to decomposeany remaining peroxide, the mixture filtered and evaporated to drynessunder reduced pressure to afford N,N-dibenzoyl (i)(cis-l,2-epoxypropyl)phosphondiamide. Inorganic salts are separated byextracting the phosphondiamide into methanol. When N,N-diethyl-cis-propenylphosphonoamide andN,N'-tetramethylcis-propenylphosphondiamide is expoxidized using thesame procedures, racemic N,N-dimethyl (cis-l,2-epoxypropyl)phosphonamide and racemic N,N,N'N'-tetramethyl-(cis-l,2-epoxypropyl)phosphondiamide is epoxidized using the sameprocedures, racemic N,N-dimethyl (cis-1,2-epoxypropyl)phosphonamide andracemic N,N,N'N'-tetramethyl- (cis-l,2-epoxypropyl)phosphondiamide areobtained respectively (Cis-l,2-epoxypropyl)phosphonic acid and its saltsare useful antimicrobial agents, which are active in inhibiting thegrowth of both gram-positive and gram-negative pathogenic bacteria. Thisantibiotic, and particularly its salts, are active against Bacillus,Escherichia, Staphylococci, Salmonella and Proteus pathogens, andantibiotic-resistant strains thereof. 11- lustrative of such Pathogensare Bacillus subtilis, Escherichia coli, Salmonella schottmuelleri,Salmonella gallinarum, Salmonella pullorum, Proteus vulgaris, Proteusmirabilis, Proteus morganii, Staphylococcus aureus and Staphyloccuspyrogenes. Thus, (cis-l,Z-epoxypropyUphosphonic acid and salts thereofcan be used as antiseptic agents to remove susceptible organisms frompharmaceutical, dental and medical equipment and other areas subject toinfection by such organisms. Similarly, they can be used to separatecertain micro-organisms from mixtures of micro-organisms. Salts of(cis-1,2- epoxypropyhphosphonic acid are also useful in the treatment ofdiseases caused by bacterial infections in man and animals and areparticularly valuable in this respect, since they are active againstresistant strains of pathogens. These salts are especially valuable,since, they are effective when given orally, although they can also beadministered parenterally.

Since the antibiotic and its salts are very active in inhibiting thegrowth of various species of Salmonella, it can be used as adisinfectant in washing eggs and areas subject to infection bySalmonella. The salts of (cis-l,2-epoxypropyl)phosphonic acid are alsouseful as bactericides in various industrial applications, for example,in inhibiting undesirable bacterial growth in the white water in papermills and in paints such as polyvinyl acetate latex paint.

When (cis-l,Z-epoxypropyl)phosphonic acid or its salts are used forcombatting bacteria in man or lower animals, they may be administeredorally in a dosage unit form such as capsules or tablets, or a liquidsolution or suspension. Alternatively, the antibiotic can beadministered parenterally by injection. These formulations can beprepared using suitable diluents, extenders, granulating agents,preservatives, binders, flavoring agents, and coating agents known tothose skilled in this art.

(cis-l,Z-epoxypropyl)phosphonic 'acid can be represented by the formula:

OH H H g/ H3o o--o- This substance is an acidic compound which is namedas (cis-1,Z-epoxypropyl)phosphonic acid in accordance with presentchemical nomenclature practice; the indicating that this phosphonic acidrotates plane-polarized light in a counterclockwise direction (to theleft as viewed by the observer) when the rotation of its disodium saltis measured in water (5 percent concentration) at 405 mu. Thedesignation cis used in describing the l,2-epoxypropylphosphonic acidcompound means that the hydrogen atoms attached to carbon atoms 1' and 2of the propylphosphonic acid are the same side of the oxide ring.

The structural formula of this antibiotic substance has been shown inthe planar formula for the sake of convenience. However, the antibioticcan also be depicted spatially as follows:

O OH

The dextrorotatory enantiomer of (cis-l,2-epoxypropyl)phosphonic acidcan be converted to cis-propenylphosphonic acid by heating withpotassium thiocyanate in aqueous methanol. The cis-propenylphosphonicacid thus obtained can be used as a starting material in the processesdescribed herein-to produce the levorotatory enantiomer of (cis-l,Z-epoxypropyl)phosphonic acid.

What is claimed is:

The process for preparing (cis-l,2-epoxypropyl)phosphonic acid whichcomprises subjecting an enantiomeric mixture of esters and amides of(cis-l,2-epoxypropyl)phosphonic acid to the action of the enzymesproduced by growing a micro-organism capable of selectively hydrolyzingan ester or amide of the isomer.

2. The process of claim 1 wherein the ester is a monoester of theformula or salts thereof wherein R is a lower alkyl group of one toseven carbon atoms, a lower alkenyl group of two to seven carbon atoms,or a lower alkynyl group of two to seven carbon atoms.

3. The process of claim 1 wherein the amide is of the formu- O NRlRgwherein Z is --NR,R or OH R, and R represent a hydrocarbyl group, asubstituted hydrocarbyl group or hydrogen and can be the same ordifferent.

4. The process of claim 1 wherein the micro-organism is a memberAspergillus. Fungi imperfecti group of micro-organisms.

5. The process of claim 1 wherein the micro-organism is a Penicillium.

6. The process of claim 1 wherein the micro-organism is an aspergillus.

7. The process of claim 1 wherein the micro-organism is anActinomycetes.

8. The process for preparing (cis-l,2-epoxypropyl)phosphonic acid whichcomprises subjecting an enantiomeric mixture of esters or amides of(cis-l,2-epoxypropyl)phosphonic acid to the action of the enzymesproduced by growing a micro-organism capable of selectively hydrolyzingan ester or amide of the isomer, to (cis l,2-epoxypropyl)phosphonicacid, and separating and recovering said acid in the form of a saltthereof.

9. The process of claim 8 wherein the micro-organism is Fungiimperfecti.

10. The process of claim 8 wherein the microorganism is a Penicillium.

11. The process of claim 8 wherein the micro-organism is an Aspergillus.

12. The process of claim 8 wherein the micro-organism is anActinomycetes.

UNITED STATES PATENT OFFICE @ETHQME or QEQ'HN a n 3,635,795 DatedJanuarv 1L8, 1.972

Inventor(s) Arnold L, Demain, et al I It is certified that error appearsin the above-identified patent and that said Letters Patent are herebycorrected as shown below:

Column 10, line 12, "and" should be or line 40, "Aspergillus" should beof the 2 line 45, "aspergillus" should read Asperqilllgs line 56,, after"is", insert Signed and sealed this 17th day of October 1972.D

(SEAL) Attest:

EDWARD M.FLETCHER,JR., ROBERT GO'ITSCHALK Attesting Officer Commissionerof Patents po'wso (0459) uscoMM-oc 60376-P69 U.S, GOVERNMENT PRINTINGOFFICE: I955 0-366-334.

2. The process of claim 1 wherein the ester is a monoester of theformula or salts thereof wherein R is a lower alkyl group of one toseven carbon atoms, a lower alkenyl group of two to seven carbon atoms,or a lower alkynyl group of two to seven carbon atoms.
 3. The process ofclaim 1 wherein the amide is of the formula wherein Z is -NR1R2 or OH R1and R2 represent a hydrocarbyl group, a substituted hydrocarbyl group orhydrogen and can be the same or different.
 4. The process of claim 1wherein the micro-organism is a member Aspergillus. Fungi imperfectigroup of micro-organisms.
 5. The process of claim 1 wherein themicro-organism is a Penicillium.
 6. The process of claim 1 wherein themicro-organism is an aspergillus.
 7. The process of claim 1 wherein themicro-organism is an Actinomycetes.
 8. The process for preparing (-)(cis-1,2-epoxypropyl)phosphonic acid which comprises subjecting anenantiomeric mixture of esters or amides of(cis-1,2-epoxypropyl)phosphonic acid to the action of the enzymesproduced by growing a micro-organism capable of selectively hydrolyzingan ester or amide of the (-) isomer, to (-)(cis-1,2-epoxypropyl)phosphonic acid, and separating and recovering saidacid in the form of a salt thereof.
 9. The process of claim 8 whereinthe micro-organism is Fungi imperfecti.
 10. The process of claim 8wherein the micro-organism is a Penicillium.
 11. The process of claim 8wherein the micro-organism is an Aspergillus.
 12. The process of claim 8wherein the micro-organism is an Actinomycetes.